Method of obtaining a two phase system of artificial immiscibility



METHOD OF OBTAINING A TWO P i in DE ARTIFICIAL I il a Michel Comar, Clanea, Morocco No Drawing. Application Feb 17 1955 I Serial No. 489,001

Claims priority, application France February 2, 1954 6 Claims. (Cl. 252-364) ,for example to the purification of organic substances and which consists in introducing this substance with its impurities, or this mixture of substances, under the proper physical conditions (pH, temperature, etc.) for ensuring their separation, into a two phase liquid system which itselflis obtained by putting together two liquids which are miscible with each other and a solid which is soluble in one of them and insoluble in the other, or into one phase of this system, which is subsequently placed in contact with the other phase.

The present invention has for an object to provide a relatively convenient method by which the constituents of the two-phase system can be qualitatively and quantitatively determined in practice, a subsidiary object being to determine more precisely the useful range of application of this process.

For this purpose, the above general principle will be illustrated by various examples of fractionation of substances having similar physical, chemical or physicochemical characteristics, these examples having been chosen from widely diverse branches of chemistry and more particularly amongst those concerning the separation of substances which are water-soluble either by nature or by virtue of the conditions in which they are, these substances being present either in the state of substances already purified or pure or in the form of raw material from which the first fractionations are to be carried out. 4

For each of these examples there will first be formulated the general (overall) composition of the selected artificial immiscibility system so arranged as to obtain optimum equilibrium between the two phases, this optimum being confirmed by tests in the course of which any volume of one phase is placed together with any desired volume of the other phase without alteration of the relative volumes after agitation and settling. In all those examples the relation between parts in weight and in volume will be that of grams and cubic centimetres.

In order to obtain such a system of immiscible phases (this immiscibility being the feature which such a system must possess for counter-current operation), one proceeds according to the invention in the following manner: decreasing quantities of a concentrated solution of the solid in the dissolving liquid (for example water and a ,ESWJ? Patted as. 31, 1959 ing liquid, and finally the various volumes of the nondissolving liquid; and from this one vdeducts the mean value from which one can ascertain by calculation the relative proportions of the solid and .of the two liquids in a manner of which an example will be given further below:

Similarly one repeats the same operations, utilising mixtures with inversely increasing and decreasing proportions of the two liquids, while introducing into these various mixtures a concentrated solution of the solid,

until one obtains, as above, equality of thephases for each of these mixtures.

By calculating oncemore a mean value amongst the various results, one obtains finally a new proportion between the three constituents of the artificial immiscibility system. One establishes then the mean value of the first and second relation and obtains a formula which in most cases willsatisfy the tests of immiscibility.

All that has been said up to now is obviously only true when these operations are carried out inside the two limits of possibility, namely beginning with a maximum concentration of solid not leading to crystallisation of the latter by the addition of the non-dissolving liquid, and on the .other handto a minimum concentration of solid in which the equality of phases, here required as test, is no longer obtainable. This definition of the two limits is essential. It has been found that one obtains maximum precision if one first determines with maximum precision these two extreme limits. Naturally it is desirable to multiply the number of measurements between these two limits, but

it remains possible to carry out the analysis described by knowing that the optimum is already approximately defined by the test haliwaybetween these two limits.

The following table, which is set up for the case of the system isopropyl alcohol-water-sodium sulphate, records the results obtained in these two series of operations and the two primary mean values from which the optimum mean value is drawn.

In these tables, it will be noted that those equalibria at which crystallisation of the solid takes place must not be retained; when crystallisation no longer takes place, one enters into the real possibilities'of an artificialimmiscibility system; similarly on the other hand progres-,

sive addition of the liquid which dissolves the solid, and which is miscible with the other liquid, will lead eventually to the impossibility of obtaining equality of the phases, which is assumed to be the chosen type of equilibrium.

EXAMPLE A System sodium sulphate-water-isopropanol at 35 C.

Y concentrated solution:

water-soluble salt) are diluted with increasing quantities 65 Na,so,, 34.10 g.; water, 91 cm=100 cm; at 35 c.

aeaogwo 4 Tab ii volumes are in the proportion 5:1. This proves that the two phases are mutually immiscible. mopmpanol 50mm of Under these conditions one ascettains by analysis the SOtNai composition of each of the phases and is then able to 5 reconstitute these phases separately in order to carry 9 7 Crystals out operations suchas extraction, purification, etc. g if 12:32 A very great variety of artificial immiscibility systems M M -8 can be produced. It has already been observed that the A 3 possibilities of artificial immiscibility with water and 6 4 8 10 water miscible liquids were the greater as the number of 2- carbon atoms of said liquids was higher. g 2 Similarly it appears that in general the possibilities of as humble the immiscibility between water andvwater miscible sol- Total 6 a7 6 70 vent depended to a great extent upon the degree of afiinity for water of the solids soluble therein. But this rule is not absolute, since other considerations may intervene, for example low degree of solubility of the solid in the water miscible solvent, etc.

. In the following table marked Example B approxi- Nassot Water Impromately 70 different cases are combined. This table bears witness to the exceedingly great number of the possibilities of attaining artificial immiscibility. In this table the Constituents, the value for water being reduced to unity, I and the sulphate in dry weights.

3%; i 3 21; cases in which immiscibility has not been obtained are 0 2485 1 0-528 1 indicated byan 0, and the cases in which it has been 25 obtained are marked by a The cases in which the- EXAMPLE 3.,

FACILITY INCREASING WITH INCREASING N0. c--.

F lAtl M1 not! Pro- Iso water e l d fin alitohol A100 01 picnic Acetone 5i mo t 22 15 Acid Alfiohol t a t at X X 1 31" i 5 0 A '6' o a e o 0 E g o 0 5 5 i 28" "i inn Siam 510 i 2i o o X o o o o i 0 Verification by synthesis: by mixing 2.48 g. of Na S0 constituents enter into chemical reaction with each other 10 cc. of water, and 5.28 cc. of isopropanol, one obtains are crossed out.

two phases each of 7.4 cc.; if one removes one cc. of While the table generally has been established at orthe upper phase and adds to it 5 cc. of the lower phase, dinary temperature, in certain cases, in which immiscione will find after agitation and settling that the volumes bility occurs only at a higher temperature, this temhave a proportion 1:5. Similarly, if one vremoves 5 cc. pcrnturc has been indicated.

of the upper phase and adds to it one cc. of the lower The selection of a system of artificial tmmlsclblllty phase, one finds after agitation and settlement that the adapted to permit optimum separation of two given sub stances to be introduced into the system-canbe made on the basis of various considerations, and while the example which will be given further below, viz. that of the separation of amino acids may serve as a guide, the coefiicient of-selectivity, that is to say the ratio of the distribution coefiicients to two given substances in an artificial immiscibility system contemplated assuitable cannot be predicted a priori; moreover there exist unpredictable aflinities for substances which would appear to be close to each other, while on the other hand substances of characters which at a first glance would appear more apart, must be subjected to systematic tests in a great number of systems before a system is discovered which satisfies the requirements of a given object; preferably this object relates to the possibility of separating in one operation two substances byvtrying to find in respect of one of these a zero distribution coefiicient to the benefit of the upper phase or of the lower phase. When on the other hand the substances are extremely close, one will have to be satisfied with a simple coefficient of selectivity which however in the majority of cases will still be sufficiently favourable when the selectivity is of the order of one or two (such as is for example the case in the separation of leucine and isoleucine as indicated further below). These selectivity coeflicients have the more practical value as the distribution coeflicient of the two substances or of one of the two substances is approximately unity.

It is thus advisable to carry out a preliminary study of the distribution coeificients and for this purpose only small quantities of the substances to be separated (for example less than 2% of the overall two-phase volume submitted to the test) are introduced into the various systems taken into consideration, in order to avoid the risk of the theoretic distribution coefficients being modified by disturbing characteristics or even constituents of the substances in the chosenartificial immiscibility system. Alternatively the equilibrium will have to be studied as above, keeping count of all these constituents, whereupon the distribution coefficients can be modified, Whichwill be generally'necessary in industrial execution. An example of the modification which the substance dealt with may produce as regards the constitution of an artificial immiscibility system and thus the distribution coefiicient, in the case of saccharose, introduced into an equilibrium of calcium chloride, water, and acetone.

In such a'system the distribution coefiicient of saccharose when introduced in the above indicated small proportions is zero to the disadvantage of the upper phase, a proportion which may be represented by the portion O/N. If the quantities of saccharose are increased, the constitutional equilibrium of the system must be reconsidered while the saccharose starts to pass into the upper phase; in the extreme case the saccharose actually forms with water and acetone an artificial immisicibility in which saccharose is represented in abundant quantity in both phases, in a proportion of approximately 1 to 5.4.

Between these proportions of 1 to 5.4 and O/N, various equilibria may be obtained wherein the saccharose and the calcium chloride are present in various proportions, and as a result one can obtain the whole range of distribution coefiicients for the saccharose between In the following examples there will be formulated in succession: v t

(1) The relations between the three constituents in order to obtain equilibrium of the type which shall not be perturbed by the substances to be separated and in fact not modified when as mentioned above, these substances are introduced in weak quantities, then:

(2) The distribution coefiicients of the substances which are required to be separated, it being understood that from the point of view of the indicated systems and of the obtained distribution coefiicients the question is not necessarily that of describing optimum separation conditions of the considered substances.

EXAMPLE 1 Separation of ferric chloride and ofaluminium chloride in solution (acid reaction) With the following system:

the following distribution coeflicients were obtained at 35 C.

For the ferric chloride For the aluminium chloride ifi EXAMPLE 2 Fractionation in raw materials or vegetable and animal extracts (a) Case ofthe purification of insulin:

The extraction of pancreas by water miscible solvents produces after filtration and concentration a mixture formed of water-soluble substances (salts, organic substances, etc.), pseudo-solutions (proteins, including insulin, and related substances), and water-insoluble, substances more particularly lipides.

All these substances form amongst themselves complex associations resulting in mutual solubility the outcome of which is resistance more particularly to the elimination of the lipides, while on the other hand the elimination of the latter is indispensible in order to perv mit the further purification of the insulin by separation A from the other proteins.

The combination of a demixing operation such as water-acetone-petroleum ether, and of an artificial immiscibility system, such for example as ammonium sulphate-water-acetone, leads to a three-phase system permitting to separate jointly and at the same time the proteins and the lipides, the former distributing themselves between the central and the lower phases.

assume The principal constitution of such a system is for example as follows:

Three phases are formed, each having a volume of approximately 0.86; the upper phase contains all the lipides, and in a slightly acid medium having a pH between and 6 the proteins distribute themselves amongst the medium and lower phase in proportions of approximately 5/1.

(b) Case of the purification of substances contained in the liver tissue:

Very finely ground liver pulp is introduced into a threephase system analogous to the one mentioned above; the upper phase is of golden yellow colour and contains in particular all the lipides; in the pale green central phase and in the brown-red opalescent lower phase those substances are distributed which after purification will become the purified liver extracts.

(0) Case of vegetable pulp:

Into a similar three-phase system finely ground carrot pulp is introduced (or the water of this pulp is utilised as the aqueous constituent of this system).

One obtains in the upper phase all the carotene while all the insoluble residues are found placed between the central and lower phases (this is also the case in the two 1 preceding cases) chlorophyll from leaf 8 (b) Case ot' the separation of two homologues which are only distinguished by a methylene group, which however is located in the main structure (armature), spacing in practice two principal carboxyl groups; viz: aspartic demixing maximum efficiency in one single operation tions. Moreover by giving the various constituents of a starting material each its proper place in one of the phases acid and glutamic acid.

Using the above'mentioned system but at pH 3.2, the distribution coeflicients are as follows:

Aspartic acid O/N Glutamic acid l/7.2

(c) Case of a closer relationship: glycocoll and alanine; in this case the difference of constitution relates to a CH; group in a side chain.

(d) Case of an extremely close relationship; isomery relating to a structural detail of a lateral chain: leucine and isoleucine.

When using the system Na co 1 0.250 Water 1 Isopropyl alcohol 0.561

at C(and in a constitutionally alkaline medium, the distribution cocfilcients are as follows:

Leucine Isoleucine (2) By the following example it may moreover be shown byway of general indications that in the majority of the hitherto studied cases the distribution coeflicients vary progressively along the whole of a given chemical by reason of their respective afiinity for the different phases, one avoids the usual retaining etfects which are detrimental to the output and industrial economy and due to the parasitic gluey masses of the extractions of hiological raw materials. Finally the upper phase is no longer contaminated by residues of starting materials or distillation residues, since these place themselves between the central and lower phases.

EXAMPLE 3 Separation of amino acids Due to the fact of their close structural relationship series. Thus in a balanced system constituted by KCl (0.24), water (1), and isopropanol (0.91) the following distribution coeflicients have been found at 45 C.:

. Clycocoll 1/77 Alanine 1/20 Valine 1/4.6 Isoleucine 1/2 Leucine 1/1,7

and the fact that their principal chemical functions are three principal groups and the other in respect of the replacement of a guanidine group by an amino group.

When using the system M 80 crystal 0.50 Water 1 Isopropyl alcohol 0.62

at 30 C. in an ammoniacal medium, approximately N/ 10, the distribution coefiicients are as follows:

Lysine Arginine EXAMPLE 4 Separation of two alkaloids of a similar isoquinolelnic constitution, obtained from a starting material (hydrastis extract): berberine and hydras'tine in the state of sulphates at 45 C. and in a sulphuric medium approximately N110, the distribution coeflicients are:

Berbcrine sulphate 13/1 Hydrastine sulphate 4/1 EXAMPLE 5 Separation of two glucosides from their starting substances (aloe): barbaloin and isobarbaloin The aloe is introduced into the following system at 35' v C., vlz:

NaCl 0.20 Water Acetone The aloe dissolves totally (apart from some brownish flakes which remain at the surface separating the two phases) and becomes distributed amongst the two phases each of which assumes a dark colour butof difierent shades; the distribution coefiicieut is as follows:

Barbaloin Approx. 2.7/1 Isobarbaloin Approx. 1/ 1 EXAMPLE 6 Extraction of a hormone: adrenalin from an extract of suprarenal glands The liquid obtained by maceration of suprarenal glands with water and water-miscible solvent in an acid medium is filtered and concentrated; the concentrate is introduced into the following system:

MgSO; Water 1 Isopropyl alcohol 0.62

the distribution coeificient at 30 C. and pH 5.8 is l/ 2.5.

EXAMPLE Vitamins B and B;

Using the system MgSO, water-isopropyl alcohol as above at 30 C. and pH 3.8, the distribution coeflieients are as follows;

For vitamin B, 1/1.4 For vitamin B2 N/O (traces) liquid in which the solid is insoluble can be used, pro-- vided its phases-have been found to be immiscible with each other in the sense of not changing their relative quantities when added to each other in different proportions and allowed to settle.

What I claim is: v

1. A method of providing a two-phase liquid system of artificial immiscibility by selecting as the constituents for said system a normally solid solute, a first liquid in which solid solute is soluble, a second liquid which is miscible with said first liquid but in which said solute is insoluble, preparing a plurality of mixtures of different proportions of a first one and a second one of said constituents not being mixtures of said solute with said second liquid, adding to each of said mixtures so much of the third one of said constituents as to produce a preliminary two-phase system in which the quantities of the two-phases have a predetermined relation and preparing said two-phase liquid system of artificial immiscibility by mixing said first liquid, said second liquid, and said solute in relative proportions which at least approximately correspond'to the mean value of their relative proportions in those of the preliminary two-phase systems in which no precipitation of the solute has been observed, so that the two-phases of said system of artificial immiscibility can be used as selective solvents for the separation of substances.

2. A method of obtaining a two-phase liquid system of artificial immiscibility the two phases of which are suitable for use as selective solvents in the separation of substances, comprising selecting a solid, a first liquid in which said solid is soluble, a second liquid which is miscible with said first liquid but in which said solid is insoluble, preparing a plurality of solutions of difierent proportions of said solid in said first liquid, adding to each of said solutions so much of said second liquid as ill to produce where possible a preliminary two-phase system in which the quantities of the two phases have a predetermined relation and preparing said two-phase liquid system of artificial immiscibility by mixing said first liquid, said second liquid, and said solid 'in relative proportions which at least approximately correspond to the mean value of their relative proportions in those of said preliminary two-phase systems in which no precipitationof the solid has been observed.

3. A method of obtaining a two-phase liquid system of artificial immiscibility the two phases of which are suitable for use as selective solvents in the separation of substances, comprising selecting a solid, 2. first liquid in which said solid is soluble, a second liquid which is miscible with said first liquid but in which said solid is insoluble, preparing a plurality of mixtures of said first liquid. with diiferent proportions of said second liquid, adding to each of said mixtures so much of said solid as to produce where possible a preliminary two-phase system in whichthe quantities of the two phases have a predetermined relation and preparing said two-phase liquid system by mixing said first liquid, said second liquid, and said solid in relative proportions which at least approximately correspond to the mean value of their relative proportions in those of said preliminary two phase systems in which no precipitation of the solid has been observed.

4. A method of obtaining a two-phase liquid system of artificial immiscibility the two phases of which are suitable for use as selective solvents in the separation of" substances, comprising selecting a normally, solid solute, a

first liquid in which said solute is soluble, a second liquid which is miscible with said first liquid but in which said solute is insoluble, and a standard relation of phase quantities, preparing a plurality of solutions of different proportions of said solute in said first liquid, adding to each of said solutions so much of said second liquid as to-pro- 'duce where possible a first preliminary two-phase system in which the quantities of the two phases have said standard relation, determining afirst mean value of the relative proportion of the first liquid, the solute and the second liquid in those first preliminary two phase systems in which no crystallisation of the solid is observed, pre-- paring a plurality of mixtures of said first liquid with different proportions of said second liquid, adding to each of said mixtures so much of said solute as to produce a where possible a second preliminary two-phase system standard relation, determining a second mean value of the relative proportions of the first liquid, the solute and the second liquid in those second preliminary two-phase systems in which no crystallisation of the solute is observed and preparing said two-phase liquid system by mixing said first liquid, said second liquid, and said solute in relative proportions which at least approximately correspond to the arithmetic mean of said first and second mean values.

5. A method as claimed in claim 1, wherein said predetermined relation is equality of thephases.

6. A method of obtaining a multi-phase liquid system of artificial immiscibility the phases of which are suitable for use as selective solvents in the separation of substances, comprising selecting a normally solid. solute, a

first liquid in which said solute is soluble, a plurality of second liquids which are miscible with said first liquid but not miscible with each other, and in which said solute is insoluble, and a standard relation of phase quantities, preparing a plurality of solutions of diflt'erent proportions of said solute in said first liquid, adding to each of said solutions so much of said second liquids as to produce where possible a preliminary multi-phase system in which the quantities of the phases have said standard relation, and preparing said multi-phase liquid system by mixing said first liquid, said second liquids, and said solute in" relative proportions which at least approximately correspond to I said preliminary multi-phase systems in which no crystal- OTHER REFERENCES hsation of the solute and no chemical reactions between Weissberger: Tech. of 0m Chem VOL In, part L 2nd the constituents have been observed. ed" 301412 Interscience (1956) h Cited in the file of this patent 5 zggggtlhlglzgxand Eng. Chem, vol. 41, No. 12, pp. UNITED STATES PATENTS Laland et al.:' Acta Medica Scandinai/ica, vol. 88, 1936, 2,307,953 Potter Jan. 12, 1943 pp 620 to 2,316,7 19 Russel Apr. 13, 1943 Smith: Proc. of the Biochem. Soc., October 1948, pp. 2,423,291 Borglin July 1, 1947 10 VIII and IX. 1 

1. A METHOD OF PROVIDING A TWO-PHASE LIQUID SYSTEM OF ARTIFICIAL IMMISCIBILITY BY SELECTING AS THE CONSTITUENTS FOR SAID SYSTEM A NORMALLY SOLID SOLUTE, A FIRST LIQUID IN WHICH SOLID SOLUTE IS SOLUBLE, A SECOND LIQUID WHICH IS MISCIBLE WITH SAID FIRST LIQUID BUT IN WHICH SAID SOLUTE IS INSOLUBLE, PREPARING A PLURALITY OF MIXTURES OF DIFFERENT PROPORTIONS OF A FIRST ONE AND A SECOND ONE OF SAID CONSTITUENTS NOT BEING MIXTURES OF SAID SOLUTE WITH SAID SECOND LIQUID, ADDING TO EACH OF SAID MIXTURES SO MUCH OF THE THIRD ONE OF SAID CONSTITUENTS AS TO PRODUCE A PRELIMINARY TWO-PHASE SYSTEM IN WHICH THE QUANTITIES OF THE TWO-PHASES HAVE A PREDETERMINED RELATION AND PREPARING SAID TWO-PHASE LIQUID SYSTEM OF ARTIFICIAL IMMISCIBILITY BY MIXING SAID FIRST LIQUID, SAID SECOND LIQUID, AND SAID SOLUTE IN RELATIVE PROPORTIONS WHICH AT LEAST APPROXIMATELY CORRESPOND TO THE MEAN VALUE OF THEIR RELATIVE PROPORTIONS IN THOSE OF THE PRELIMINARY TWO-PHASE SYSTEMS IN WHICH NO PRECIPITATION OF THE SOLUTE HAS BEEN OBSERVED, SO THAT THE TWO-PHASES OF SAID SYSTEM OF ARTIFICIAL IMMISCIBILITY CAN BE USED AS SELECTIVE SOLVENTS FOR THE SEPARATION OF SUBSTANCES. 